ABSTRACTHere, we investigated the impact of mulberry fruit (MBF) extracts on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages, and the therapeutic efficacy of MBF diet in mice with dextran sulfate sodium (DSS)-induced acute colitis and MUC2(-/-) mice with colorectal cancer. In vitro, LPS-induced nitric oxide (NO) production was significantly inhibited by MBF extracts via suppressing the expression of proinflammatory molecules, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-β) and IL-6. Particularly, a dose-dependent inhibition on LPS-induced inflammatory responses was observed following treatment with MBF dichloromethane extract (MBF-DE), in which linoleic acid and ethyl linolenate were identified as two active compounds. Moreover, we elucidated that MBF-DE attenuated LPS-induced inflammatory responses by blocking activation of both NF-κB/p65 and pERK/MAPK pathways. In vivo, DSS-induced acute colitis was significantly ameliorated in MBF-fed mice as gauged by weight loss, colon morphology and histological damage. In addition, MBF-fed MUC2(-/-) mice displayed significant decrease in intestinal tumor and inflammation incidence compared to control diet-fed group. Overall, our results demonstrated that MBF suppressed the development of intestinal inflammation and tumorgenesis both in vitro and in vivo, and supports the potential of MBF as a therapeutic functional food for testing in human clinical trials.

f1: Mulberry fruit (MBF) extracts inhibit LPS-induced NO production without affecting cell viability in RAW 264.7 macrophage cells.Cells were treated with MBF dichloromethane extracts (MBF-DE), MBF ethyl acetate extracts (MBF-EE), MBF n-butanol extracts (MBF-BE) and MBF water extracts (MBF-WE) at different concentrations range from 25 to 200 μg/mL in the presence or absence of LPS (1 μg/ml) for 24 h. Quercetin (Q) was used as the positive control. NO production (a) and cell viability (b) were determined by the Griess and MTS assay, respectively. Data are expressed as means ± SD with at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to LPS-induced control.

Mentions:
After cells were treated with different concentrations of MBF dichloromethane extracts (MBF-DE), ethyl acetate extracts (MBF-EE), n-butanol extracts (MBF-BE) and water extracts (MBF-WE) ranging from 25 to 200 μg/ml in the presence of LPS, the production of nitric oxide (NO) was estimated by measuring the concentration of nitrite in cell culture medium using the Griess method. When compared with normally cultured cells in the absence of LPS, NO production was significantly induced in culture medium after 24 h of LPS treatment (Fig. 1a). However, the LPS-stimulated NO accumulation was significantly decreased by simultaneously adding MBF-DE, MBF-EE, MBF-BE or MBF-WE in the growth medium (Fig. 1a). Among them, MBF-DE treatment showed the highest inhibitory efficiency in NO production in a dose-dependent manner (Fig. 1a). However, the reduction of NO production in culture medium was not due to cytotoxicity, since cell viability was not affected by the treatment with MBF-DE, MBF-EE, MBF-BE or MBF-WE in the presence of LPS (Fig. 1b).

f1: Mulberry fruit (MBF) extracts inhibit LPS-induced NO production without affecting cell viability in RAW 264.7 macrophage cells.Cells were treated with MBF dichloromethane extracts (MBF-DE), MBF ethyl acetate extracts (MBF-EE), MBF n-butanol extracts (MBF-BE) and MBF water extracts (MBF-WE) at different concentrations range from 25 to 200 μg/mL in the presence or absence of LPS (1 μg/ml) for 24 h. Quercetin (Q) was used as the positive control. NO production (a) and cell viability (b) were determined by the Griess and MTS assay, respectively. Data are expressed as means ± SD with at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to LPS-induced control.

Mentions:
After cells were treated with different concentrations of MBF dichloromethane extracts (MBF-DE), ethyl acetate extracts (MBF-EE), n-butanol extracts (MBF-BE) and water extracts (MBF-WE) ranging from 25 to 200 μg/ml in the presence of LPS, the production of nitric oxide (NO) was estimated by measuring the concentration of nitrite in cell culture medium using the Griess method. When compared with normally cultured cells in the absence of LPS, NO production was significantly induced in culture medium after 24 h of LPS treatment (Fig. 1a). However, the LPS-stimulated NO accumulation was significantly decreased by simultaneously adding MBF-DE, MBF-EE, MBF-BE or MBF-WE in the growth medium (Fig. 1a). Among them, MBF-DE treatment showed the highest inhibitory efficiency in NO production in a dose-dependent manner (Fig. 1a). However, the reduction of NO production in culture medium was not due to cytotoxicity, since cell viability was not affected by the treatment with MBF-DE, MBF-EE, MBF-BE or MBF-WE in the presence of LPS (Fig. 1b).

ABSTRACTHere, we investigated the impact of mulberry fruit (MBF) extracts on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages, and the therapeutic efficacy of MBF diet in mice with dextran sulfate sodium (DSS)-induced acute colitis and MUC2(-/-) mice with colorectal cancer. In vitro, LPS-induced nitric oxide (NO) production was significantly inhibited by MBF extracts via suppressing the expression of proinflammatory molecules, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-β) and IL-6. Particularly, a dose-dependent inhibition on LPS-induced inflammatory responses was observed following treatment with MBF dichloromethane extract (MBF-DE), in which linoleic acid and ethyl linolenate were identified as two active compounds. Moreover, we elucidated that MBF-DE attenuated LPS-induced inflammatory responses by blocking activation of both NF-κB/p65 and pERK/MAPK pathways. In vivo, DSS-induced acute colitis was significantly ameliorated in MBF-fed mice as gauged by weight loss, colon morphology and histological damage. In addition, MBF-fed MUC2(-/-) mice displayed significant decrease in intestinal tumor and inflammation incidence compared to control diet-fed group. Overall, our results demonstrated that MBF suppressed the development of intestinal inflammation and tumorgenesis both in vitro and in vivo, and supports the potential of MBF as a therapeutic functional food for testing in human clinical trials.